An ever-increasing number of viruses are being identified as the source of human disease. The better known virally-caused diseases include chicken pox, measles, mumps, influenza, hepatitis, poliomyelitis, rabies, and now, of course, Acquired Immunodeficiency Syndrome (AIDS).
Human immunodeficiency virus (HIV) is the etiologic agent of AIDS. See S. Crowe and J. Mills in Basic and Clinical Immunology, 7th Ed. (D. P. Stites and A. I. Terr, Eds.), pp. 697-711 (1991) at p. 697, col. 1. A complete sequencing of the HIV genome indicates that it comprises the same overall gag-pol-env organization as other retroviruses. See L. Ratner et al., Nature 313:277 (1985) at p. 277, Abstract. The virus invades a host cell and uses the host cell's machinery to replicate itself.
HIV infects cells that have a protein called CD4 on the cell surface. CD4 serves as a receptor for the virus. Lymphoid cells susceptible to infection include CD4.sup.+ T lymphocytes, monocyte-macrophages, dendritic cells, and Langerhans cells. In addition, HIV can infect non-lymphoid microglial cells, retinal cells, colonic mucosal cells, and endothelial cells, all of which have the CD4 surface antigen. See S. Crowe and J. Mills in Basic and Clinical Immunology, 7th Ed. (D. P. Stites and A. I. Terr, Eds.), pp. 697-711 (1991 ) at p. 699, col. 1.
The monocyte-macrophage cells are probably the first cells infected by HIV. Viral replication proceeds slowly in these cells, with little cytopathology, and the cells apparently become a major reservoir for the virus. See S. Crowe and J. Mills in Basic and Clinical Immunology, 7th Ed. (D. P. Stites and A. I. Terr, Eds.), pp. 697-711 (1991) at p. 697, col 1. In contrast, when HIV invades CD4.sup.+ lymphocytes, it replicates more rapidly and, through a mechanism that is not completely understood, causes depletion of the circulating CD4.sup.+ lymphocyte population.
The detection of HIV in human peripheral blood cells is now well-documented. The first assays involved isolation and culture of the virus. See M. Popovic et al., Science 224:497 (1984) at p. 497, Abtract. However, the process takes 3-4 weeks and has low sensitivity. Subsequent assays measured anti-viral antibody produced by human immune cells that contacted the virus. See F. Barin et al., Science 228:1094 (1985) at p. 1094. While this is a faster technique, it is an indirect method of detection, measuring past exposure to the virus, not present infection. Finally, direct detection of viral DNA sequences was achieved by amplification techniques such as the Polymerase Chain Reaction (PCR). See C. Y. Ou et al., Science 239:295 (1988) at p. 295 Abstract. See also J. Bell and L. Ratnet, AIDS Res. Hum. Retrovir. 5:87 (1989) at p. 87, Abstract.
PCR was developed by K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR provides a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to a sequence on their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."
With PCR, it is possible to amplify a single copy of a specific target sequence present in genomic DNA to a level which is detectable by several different methodologies (e.g., hybridization of PCR-amplified sequences with a labelled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of .sup.32 P-labelled deoxynucleotide triphosphates, e.g., dCTP or dATP, into the amplified segment). In addition to specific sequences present in genomic DNA, any oligonucleotide sequence, including HIV sequences, can be amplified with an appropriate set of primer molecules.
Using blood cells as a source of DNA, HIV sequences have been amplified sufficiently to be detected by hybridization probes. See C. Y. Ou et al., Science 239:295 (1988) at p. 295, col 2. However, the application of this method for detecting HIV in a general population has drawbacks. Blood cells and serum contain inhibitors of PCR. See R. Higuchi in PCR Technology, Principles and Applications for DNA Amplification (H. A. Erlich, Ed.), pp. 31-38 (1989). To avoid inhibition, the nucleic acid from blood cells typically must be isolated and purified prior to amplification, a step that results in a loss of sensitivity. Furthermore, only about 1 in 10,000 CD4.sup.+ lymphocytes express viral RNA in HIV-infected individuals. See C. Y. Ou et al., Science 239:295 (1988) at p. 295, col 2. See also S. Crowe and J. Mills in Basic and Clinical Immunology, 7th Ed. (D. P. Stiles and A. I. Terr, Eds.), pp. 697-711 (1991). The concentration of the HIV sequence is thus very low in comparison to total cellular sequences and low copy number is associated with additional problems in the execution of the PCR technique.
For low copy number systems, such as the presence of HIV sequence in human cells, one typically needs a larger sample size (e.g., many cells) in order to be sure the sequence of interest is present in the sample. Large sample sizes, however, have more inhibitor. Furthermore, large samples are not readily amenable to amplification because of the expense of large sample amplification, as well as the inhibiting impact of very large amounts of nucleic acid on most amplification techniques.
The above considerations are best understood by the example of the low copy situation in the case of HIV infection. The number of HIV infected CD4.sup.+ T lymphocytes (or T4 cells) can be as low as one out of every 1,000 T4 cells, and only 30% (maximum) of the total white blood cell population are T4 cells. Therefore, for each HIV PCR beginning with 40 infected T4 cells, a minimum of 100-300 .mu.l of normal whole blood will be required. A larger volume may be needed for HIV patients due to depletion of their T4 cells. However, since PCR is normally carried out in 100 .mu.l, a volume reduction step, which allows concentration of white blood cells, may be necessary to avoid the expense of using large amounts of enzyme.
One potential problem of cell concentration steps is that the final amount of DNA obtained by the procedure may be too high for PCR to efficiently proceed. It has been shown that the amount of DNA present in 0.5 ml of normal whole blood is difficult or impossible to amplify all at once by PCR. The occurrence of DNA-dependent PCR inhibition is probably due to an excess of misprimed sites (relative to enzyme molecules), which form unproductive ternary complexes with the polymerase. This results in the accumulation of a large number of linearly or exponentially amplified non-target sequences. Since the specificity of the amplification is lost as the amount of non-target DNA is increased, the exponential accumulation of the target sequence of interest does not occur to any significant degree.
In addition to the PCR-related problems associated with amplifying HIV sequences from blood samples, there are numerous problems associated with drawing blood. Certain persons may object to the invasive aspect of venipuncture. Furthermore, it requires trained personnel to draw and process the blood and this entails additional costs. These factors and the special requirements needed for proper storage of blood make it a less than optimal test method in developing countries and remote areas.
One problem that has received considerable attention is the risk of infection to health workers and pathologists who are involved with testing blood and other biological fluids. The Occupational Safety and Health Administration (OSHA) has issued guidelines on safer handling of contaminated specimens. See Guidelines for Prevention of Transmission of HIV and HBV to Health-Care and Public Safety Workers, CDC (February 1989).
Following the onset of the AIDS epidemic, there has been a renewed desire to reduce exposure of personnel to human blood and body fluid samples. Technologists who come into contact with samples from AIDS patients are aware that an infectious virus can persist in a liquid or dried state for prolonged periods of time, possibly even at elevated temperatures. Resnick et al., JAMA 255:1887 (1986) at p. 1887, Abstract.
Thus, there remains a need for a method for detecting HIV infection with speed, sensitivity, and safety. In addition, if the method is to be useful in outlying areas, simplicity of sample collection and storage are required.